Immuno-PETE

Abstract

Methods and compositions are described herein for primer extension target enrichment of immune receptor (BCR or TCR) sequences.

What is claimed is: 
     
       1. A method for enriching from a sample a plurality of structurally different target polynucleotides, wherein individual target polynucleotides of the plurality comprise immune cell receptor V, J, and C and optionally D gene regions, the method comprising:
 a) providing a reaction mixture comprising:
 i) the plurality of structurally different target polynucleotides; and 
 ii) a plurality of immune cell receptor C gene specific primers, wherein the plurality of immune cell receptor C gene specific primers having the following regions from 5′ to 3′: [5′-Phos], [SPLINT], [BARCODE], and 
 
 [FW], wherein: 
 [5′-Phos] comprises a 5′ phosphate; 
 [SPLINT] comprises an adaptor hybridization site of 2-8 nucleotides in length; 
 [BARCODE] comprises a barcode region of at least 6 nucleotides in length, wherein each nucleotide of the barcode region is independently selected from the group consisting of N and W; and 
 [FW] of each immune cell receptor C gene specific primer comprises a structurally distinct region that specifically hybridizes to an immune cell receptor C gene, 
 wherein the immune cell receptor C gene specific primers are hybridized to the C gene regions of the target polynucleotides; 
 b) extending the hybridized immune cell receptor C gene specific primers with a polymerase, and then removing un-extended immune cell receptor C gene specific primers, if present, wherein the extended immune cell receptor C gene specific primers comprise at least a portion of the immune cell receptor C region, optionally the immune cell receptor D region, at least a portion of the immune cell receptor J region, and at least a portion of the immune cell receptor V region; 
 c) hybridizing a first universal adaptor to the [SPLINT] adaptor hybridization site of the extended immune cell receptor C gene specific primers, wherein said first universal adaptor is a double-stranded adaptor comprising a single-stranded overhang region that hybridizes to the [SPLINT] adaptor hybridization site of said extended primers; 
 d) ligating the hybridized first universal adapters to the extended immune cell receptor C gene specific primers, and then removing un-ligated adapters, if present; 
 e) hybridizing a plurality of immune cell receptor V gene specific primers to the V region portions of the extended immune cell receptor C gene specific primers, wherein the immune cell receptor V gene specific primers comprise a 3′ V gene hybridizing region and a 5′ second universal adapter region; and 
 f) extending the hybridized immune cell receptor V gene specific primers with a polymerase, thereby forming a plurality of structurally different double-stranded products, each comprising at least a portion of the immune cell receptor V region, optionally the immune cell receptor D region, and at least a portion of the immune cell C region flanked by a first and second universal adapter sequence. 
 
     
     
       2. The method of  claim 1 , wherein e) and f) are repeated 2 to 15 times by heating to denature double-stranded products, cooling to hybridize un-extended immune cell receptor V gene specific primers to the V region portions of the extended immune cell receptor V gene specific primers, and extending hybridized primers. 
     
     
       3. The method of  claim 1 , wherein the removing un-extended immune cell receptor C gene specific primers comprises digesting single-stranded DNA by exonuclease digestion. 
     
     
       4. The method of  claim 2 , wherein the method further comprises amplifying double-stranded products comprising first and second universal adapters by universal PCR. 
     
     
       5. The method of  claim 4  wherein the method further comprises determining an isotype or clonotype of at least one of the plurality of structurally different double-stranded products or amplified double-stranded products. 
     
     
       6. The method of  claim 1 , wherein the [FW] of each immune cell receptor C gene specific primer specifically hybridizes to a framework 1, framework 2, or framework 3 region of a T cell receptor C gene. 
     
     
       7. The method of  claim 1 , wherein the [FW] of each immune cell receptor C gene specific primer specifically hybridizes to a framework 1, framework 2, or framework 3 region of a B cell receptor C gene. 
     
     
       8. The method of  claim 1 , wherein the plurality of immune cell receptor C gene specific primers comprises at least 2 of the primers set forth in SEQ ID Nos: 205-213. 
     
     
       9. The method of  claim 1 , wherein the [SPLINT] consists of 6 consecutive nucleotides. 
     
     
       10. The method of  claim 1 , wherein the [BARCODE] consists of thirteen consecutive nucleotides selected from the group consisting of N and W. 
     
     
       11. The method of  claim 1 , wherein the plurality of immune cell receptor C gene specific primers comprises at least 5 of the primers set forth in SEQ ID NOs: 205-213.